Polymer Composite Nanomaterial Encapsulation System

Generally, a nanomaterial polymer encapsulation system useful in the production of nanocomposites comprising a hydrophobic nanoparticle encapsulated in a hydrophobic region of a polymer with the external hydrophilic region of the polymer ensuring water-solubility and affording a functional group which can be utilized for the production of nanocomposite conjugates.

Specifically, particular embodiments can comprise nanocomposites including one or more nanoparticles including a quantum dot (“QD”) and/or a superparamagnetic iron oxide nanoparticle (“SPION”) and/or an upconverting nanoparticle encapsulated in a polystyrene-b-polyethylene glycol amine (“PS-b-PEG-NH”) affording an amine functional group that can be activated to conjugate antibodies, modified antibodies, or antibody fragments, and in a particular embodiments, activated with methyltetrazine glycol-4-N-hydroxysuccinimide esters (“methyltetrazine-PEG4-NHS ester”) to allow conjugation of trans-cyclooctyne (“TCO”) modified antibody or antibody fragments.

Nanocomposites have a diverse variety of potential applications including, but not limited to, medicine, biomedicine, biotechnology, biomaterials, biomechanics, and energy production. Current methods of polymer encapsulation of nanoparticles such as QD or SPION to produce nanocomposites have led to agglomeration of the nanocomposites limiting production to small batches, low photochemical stability, lack of uniform size and brightness, a lack of specificity of nanocomposite conjugates to cellular targets, and the lack of methodologies for rapid purification of nanocomposites bound to cellular targets. There would be substantial advantages in nanocomposites and methods of making and using nanocomposites that minimize agglomeration in large batch production, have high photochemical stability, uniform and narrow size distribution and brightness, high binding specificity to cellular targets, and protocols for rapid purification of nanocomposites bound to cellular targets.

A broad object of particular embodiments of the invention can be to provide a nanocomposite comprising one or more nanoparticles encapsulated by a polymer having a hydrophobic region associated with the nanoparticle and a hydrophilic region including a functional group associated with the aqueous environment, wherein the nanoparticle can comprise one or more of a QD or a SPION, and combinations thereof, and the polymer can comprise a polystyrene-b-polyethylene glycol including a functional group, wherein polystyrene can have a molecular weight occurring in the range of about 1.5 kDa to about 40 kDa, and wherein the polyethylene glycol can have a molecular weight occurring in the range of about 10 kDa to about 40 kDa, whereby combinations and permutations of the QD, SPION, molecular weight of the polystyrene and/or the molecular weight of the polyethylene glycol and selection of the branched structure of the polyethylene glycol, and variation in mass ratios thereof, allow for a numerous and wide variety of nanocomposites to be produced having substantially uniform hydrodynamic diameter occurring in a range of about 40 nm to about 500 nm and brightness due to QD having different emission wavelengths occurring in the range of 420 nm and 1000 nm.

Another broad object of particular embodiments of the invention can be to provide a QD and/or SPION nanocomposite antibody conjugate capable of specifically binding a cellular target, wherein illustrative embodiments of the nanocomposite antibody conjugate include one or more of anti-CD3 or anti-CD4 antibodies capable of specifically binding CD3 and or CD4 peripheral blood mononuclear cells.

Another broad object of particular embodiments of the invention can be to provide nanocomposites comprising polymer encapsulated QD or SPION, or combinations thereof, for specific targeting of macrophages, wherein illustrative embodiments comprise the uptake of embodiments of nanocomposites by hemocytes.

Another broad object of particular embodiments of the invention can be to provide a method of isolating QD and/or SPION nanocomposite antibody conjugates bound to cells, wherein SPION nanocomposite antibody conjugates bound to the cells can be separated and isolated by influence of a magnetic field, and wherein isolated QD or SPION nanocomposite antibody conjugates bound to cells can be analyzed by flow cytometry, and in particular embodiments the analyzed QD or SPION nanocomposite antibody conjugates bound to the cells can be flow sorted into discrete populations based on one or more characteristics of the cells.

Naturally, further objects of the invention are disclosed throughout other areas of the specification, drawings, photographs, and claims.

The present invention may be understood by reference to the following detailed description of aspects of the invention and the examples included therein and to the figures and their previous and following description. Compounds, compositions, articles, devices, or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments for the purpose of enabling a person of ordinary skill in the art to make and use a numerous and wide variety of embodiments of the invention, even if not explicitly disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which may need to be independently confirmed.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component,” “a polymer,” or “a particle” includes mixtures of two or more such components, polymers, or particles, and the like.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, as illustrative examples, an ethylene glycol residue in a polyester refers to one or more —OCHCHO— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more —CO(CH)CO— moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, and groups of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, elements in methods of making and using the compositions of the invention. Thus, if there are a variety of additional elements that can be performed it is understood that each of these additional elements can be performed with any specific embodiment or combination of embodiments of the methods of the invention. It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

Now, with primary reference to, a block flow diagram provides a general overview of the nanomaterial polymer encapsulation system () including one or more of: polymer synthesis (Block 1A) of a nanoparticle (“NP”) () encapsulation polymer (“P”) (); formation of polymer encapsulated nanoparticles (“PENP”) () (Block 1B); preparation of antibodies, half-antibodies, antibody fragments (individually or collectively “Ab”) () (Block 1C); preparation of PENP antibody conjugates (“PENP-Ab”) () (Block 1D); PENP-Ab cell labeling (PENP-Ab-Cells”) (), flow cytometry analysis () and imaging of PENP-Ab-Cells () (Block 1E).

Now, with primary reference to, a block flow diagram provides an overview of an illustrative polymer synthesis process of, Block 1A. Referring to Block 2A of the process, aminopolystyrene (“PS-NH) having linear formula (CH)CHN, wherein the PS molecular weight can occur in a range of about 1.5 kDa to about 40 kDa, can be obtained from Polymer Source, Inc., PN P3965-SNH, comprising Formula I. Particular embodiments can include PS-NHhaving a number averaged molecular weight of about 9.5 kDa.

Now, referring to, Block 2B of the polymer synthesis process, dibenzocylooctyne-N-hydroxysuccinimidyl ester (“DBCO-NHS Ester”) having liner formula CHNO(CAS No.: 1353016-71-3) and having a molecular weight of 402.40 g/mol can be obtained from Click Chemistry Tools, PN A133-100 comprising Formula II.

Now, referring to, Block 2C of the polymer synthesis process, PS-NHcan be reacted with DBCO-NHS Ester to produce polystyrene-dibenzocylooctyne (“PS-DBCO”) comprising Formula III.

An example of a scalable procedure for the production of PS-DBCO can include thawing PS-NH20 mg for 15 minutes at room temperature (“RT”) and thawing DBCO-NHS-Ester 10 mg for 15 minutes (“min.”) at RT. Aliquot 1 mL of toluene (CHCH) (CAS No.: 108-88-3) to the PS-NH20 mg and vortex for 15 minutes at 500 RPM. Centrifuge DBCO at 1500 relative centrifugal force (“RCF”) (RCF=(RPM)×1.118×10×r) for 30 seconds (“sec.”) at about 25° C. (about 77° F.). Aliquot 1 mL of toluene to the DBCO. Sonicate the DBCO for 5 min. in RT water. Transfer 1 mL of DBCO in toluene to PS-NHin toluene. Vortex DBCO in PS-NHmixture for 16 to 24 hours (“hr.”) at 500 RPM at RT.

Now, referring to, Block 2D of the polymer synthesis process, an 8-arm polyethylene glycol-amine (“PEG-NH”) comprising a multi-arm PEG derivative with amine groups at each terminal of the eight arms connected to one hexaglycerol core having linear formula R(O(CHCHO)nCHCHNH)and a number averaged molecular weight of about 19.5 kDa can be obtained from Nanosoft Polymers, PN 2443, comprising Formula IV and further shown in Formula V.

In particular embodiments, a 4-arm polyethylene glycol-amine, a 6-arm polyethylene glycol-amine, or an 8-armpolyethyleneglycol-amine, or combinations thereof, can also be utilized having corresponding PEG molecular weights of 10 kDa, 20 kDa, or 40 kDa.

Now, referring to, Block 2E of the polymer synthesis process, an azido-d-polyethylene glycol 4-N-hydroxysuccinimidyl ester (“Azido-PEG-NHS Ester”) having a molecular weight of 388.37 g/mol (CAS No.: 944251-24-5) can be obtained from Click Chemistry tools PN AZ103-100 comprising Formula VI.

Now, referring to, Block 2F of the polymer synthesis process, PEG-NHcan be reacted with Azido-PEG-NHS Ester to produce branched polyethylene glycol (“PEG-Amine-Azide”) comprising Formula VII.

An example of a scalable protocol for the production of PEG-Amine-Azide can include thawing 40 mg of PEG-NHfor 15 min. at RT and thawing 80 μl of Azido-PEG-NHS Ester (50 mM) for 15 min. at RT. Aliquot 400 μl of methanol (CAS NO.: 6756-1) to PEG-NHand vortex the PEG-NHfor 15 min. at 500 RPM. Centrifuge Azido-PEG-NHS at 1500 RCF for 30 sec. at 25° C. (about 77° F.). Transfer the Azido-PEG-NHS 80 μl to the 400 μl of PEG-NH. Vortex for 16 to 24 hr. at 500 RPM at RT.

Now, referring to, Block 2G of the polymer synthesis process, PS-DBCO obtained in Block 2C of the synthesis process can be purified by the following illustrative scalable procedure including transferring 400 μl of PS-DBCO to each of five centrifuge tubes. Aliquot 800 μl of methanol to each of the five centrifuge tubes. Mix PS-DBCO in methanol by inversion of each of the five centrifuge tubes. Centrifuge the PS-DBCO in methanol for 5 min. at 20,000 RCF at 15° C. to 25° C. (about 59° F. to about 77° F.). Decant the supernatant from each of five centrifuge tubes. Add 400 μl of toluene to each of the five centrifuge tubes. Place the five centrifuge tubes in a water bath at 37° C. (about 98.6° F.) for 2 min. Dissolve the PS-DBCO pellet in 400 μl toluene by mixing with a pipette. Add 800 μl of methanol to each of the five centrifuge tubes. Invert each centrifuge tube to mix contents. Centrifuge at 20,000 RCF for 5 min. at 15° to 25° C. (about 59° F. to about 77° F.). Decant the supernatant from each of five centrifuge tubes. Add 400 μl of toluene to each of the five centrifuge tubes. Place the five centrifuge tubes in a water bath at 37° C. (about 98.6° F.) for 2 min. Dissolve the PS-DBCO pellet in toluene by mixing with a pipette.

Now, referring to, Block 2H of the polymer synthesis process, PS-DBCO obtained in Block 2G of the synthesis process can be reacted with PEG-Amine-Azide obtained in Block 2F of the polymer synthesis process to produce polystyrene-b-poly (ethylene glycol) amine (“PS-b-PEG-NH”) comprising Formula VIII.

In the instant illustrative example of Block 2H, the PS-DBCO in toluene of all five centrifuge tubes from Block 2G and PEG-Amine-Azide in methanol obtained in Block 2F can be transferred to a 4 mL glass vial. Vortex the mixture of PS-DBCO and PEG-Amine-Azide at 500 RPM at RT for 16 to 24 hr. to produce PS-b-PEG-NHcomprising Formula VIII. The PS-b-PEG-NHcan comprise PS having molecular weights ranging from about 1.5 kDa to about 40 kDa and can comprise PEG having molecular weights ranging from about 10 kDa to about 40 kDa.

Referring to, Block 2I of the synthesis process, the PS-b-PEG-NHobtained in Block 2H can be purified and dried by the following illustrative scalable procedure including cooling hexane (CAS No.: 110-54-3) at −20° C. (about −4° F.) for 15 min. Aliquot approximately 1.25 mL of PS-b-PEG-NHobtained in Block 2I of the synthesis process into a 15 mL centrifuge tube. Slowly add 12 mL of hexane to the centrifuge tube containing PS-b-PEG-NH. Mix the PS-b-PEG-NHin toluene/hexane by gently tilting the centrifuge tube 10 to 15 times. Place the centrifuge tube at −20° C. (about −4° F.) for 5 min. to cool the PS-b-PEG-NHin toluene/hexane. Decant the supernatant from the PS-b-PEG-NHpellet. Aliquot 1 mL of tetrahydrofuran to the centrifuge tube. Place the centrifuge tube in a water bath at the 37° C. (about 98.6° F.) for 2 min. Dissolve the PS-b-PEG-NHpellet by mixing with a pipette. Slowly add 6 mL of cold hexane to the centrifuge tube. Gently tilt the centrifuge tube 10 to 15 times until PS-b-PEG-NHprecipitates as flakes are visualized with clear supernatant. Place the centrifuge tube at −20° C. (about −4° F.) for 5 min. Decant the supernatant from the PS-b-PEG-NHpellet. Place the centrifuge tube containing the PS-b-PEG-NHpellet at RT for 24 hr. to remove excess solvents and to obtain a dry PS-b-PEG-NH. Store at −20° C. (about −4° F.).

Now, referring primarily to, the structure of the PS-b-PEG-NHobtained in Block 2I of the synthesis process can be analyzed using nuclear magnetic resonance (“NMR”). The NMR spectrum shown invalidates the molecular structure obtained by the polymer synthesis process shown in, Blocks 2A through 2I, and above disclosed polymer synthesis procedure, is purified PS-b-PEG-NHcomprising formula VIII.

The illustrative example of the polymer PS-b-PEG-NHobtained in Block 2I is not intended to preclude embodiments of the PS-b-PEG including functional groups other than NH. Other functional groups can include as examples, one or more of: acrylate, maleimide, vinylsulfone, azide, biotin, carboxyl, thiol, alkyne, hydrazide, N-hydroxysuccinimide ester, and nitrophenyl carbonate, and combinations thereof, or embodiments including other similar or equivalent polymers including one or more functional groups.

Now, with primary reference towhich illustrates a particular embodiment of PENP () including one or more QD () encapsulated in a polymer (“P”) () having a hydrophobic region which can associate or coordinate with the one or more QD () and a hydrophilic region including a functional group () which can associate with an aqueous environment, and in particular embodiments, the polymer (P) () can comprise PS-b-PEG-NHobtained in Block 2I, which by solution based association can produce polymer encapsulated nanoparticles including one or more QD () (“PENP-MultiDots”) ().

Now, with primary reference to, which illustrates a particular embodiment of PENP () including one or more QD () and one or more SPION () encapsulated in a polymer (“P”) () having a hydrophobic region which can associate or coordinate with the one or more SPION () and optionally one or more QD () and a hydrophilic region including a functional group () which can associate with an aqueous environment, and in particular embodiments, the polymer (P) () can comprise PS-b-PEG-NHobtained in Block 2I, which by solution based association can produce PENP () including one or more SPION () and one or more QD () (“PENP-MagDots”) ().

Now, with primary reference to, a numerous and wide variety of agents () can be conjugated to PENP-MultiDots () and/or PENP-MagDots () using the functional group () of the polymer (), and in particular embodiments, the amine afforded by PS-b-PEG-NH. The example of PENP-MagDots () inillustrates that one or more agents () can be conjugated to PENP-MultiDots () and/or PENP-MagDots () by activating the functional group (), including as illustrative examples: one or more linkers (,,), a polyethylene glycol, a fluorescent probe, an aptamer, a vitamin, a radioactive isotope, a contrast media, a surface charge modifier, a lectin, a protein, a peptide, a cell surface receptor, a cell coat, and combinations thereof. Specifically, in particular embodiments the functional group () can be utilized to directly or indirectly through one or more linkers (,,) couple an antibody or antibody fragment (Ab) (′) to produce PENP antibody conjugates () including as illustrative examples PENP-MultiDots-Ab () and PENP-MultiDots-Ab ().

Now, with primary reference to, scalable, solution-based production of PENP () encompassed by the invention, including, but not necessarily limited to, PENP-MagDots () or PENP-MultiDots (), can be prepared by conventional self-assembly, flash nanoprecipitation (FNP) comprising rapid turbulent mixing generated by high velocity flows, as described by Yanjie Zhang, a Aaron R. Clapp,, Issue 89, 2014 “Preparation of quantum dot-embedded polymeric nanoparticles using flash nanoprecipitation”, or by rapid mixing induced by electrohydrodynamics (“EHD”): EHD mixing mediated-nanoprecipitation (“EHD-NP”). Kil Ho Lee, Guolingzi Yang, Barbara E. Wyslouzil and Jessica O. Winter,2019, 1, 4, 691-700, each incorporated by reference herein.

The illustrative EHD mixing system ofcan include a syringe () having a syringe barrel () fitted with a sliding syringe plunger (), and a syringe needle (). A syringe pump () configured drive the syringe plunger () to deliver an organic phase of water-miscible nonpolar aprotic solvents (“OP”) including solubilized (P) () and NP () whether QD () and/or SPIONS () (collectively the “inorganics”) at a predetermine volumetric flow rate into an aqueous phase (“AP”). The illustrative example of a syringe () and a syringe driver () is not intended to preclude other appliances useful in delivering the OP into the AP at a predetermined volumetric flow. The concentration of inorganics per unit volume of the OP can be about 0.1 volume to volume (“v/v”) to about 0.5 v/v. The concentration of P () to QD () and/or SPION () in the OP by mass can be about 1:1 to about 4:1. The amount of P () can be adjusted to obtain a PENP () having a substantially uniform hydrodynamic diameter (“HD”) that occurs in size range of about 40 nanometers (“nm”) to about 500 nm. In embodiments in which the QD () and/or the SPIONS () are passivated with a ligand (), the ligand mass relative to the total inorganics mass can be about 20% to 40% by mass. A ligand mass percent greater than 40% can interfere with the assembly of the PENP ().

A non-electrically conductive container () can hold the AP, typically distilled or deionized water. A positive electrode () and a negative electrode () can be introduced about 1 cm apart in the AP held by the non-electrically conductive container (). In particular embodiments the syringe needle () can, if electrically conductive, act as the positive electrode (). A voltage source () can supply a voltage (“V”) to the positive terminal () to generate an electrical field between the positive electrode () and negative electrode () in the AP. The syringe plunger () can be driven to introduce the OP into the AP at a consistent flow rate of about 8 mL hto about 15 mL h. Voltage (V) can be adjusted between about-1 kilovolt (“kV”) and about-2.5 kV. The electric field can generate a fine dispersion of OP in the AP to produce PENP (), PENP-MagDots (), or PENP-MultiDots () of substantially uniform size. The resulting size of the PENP (), PENP-MagDots (), or PENP-MultiDots () can increase with increasing V and/or concentration of inorganics per unit volume of the OP. The resulting PENP-MagDots () can be subsequently isolated by influence of a magnetic field ().

PENP-MultiDots () can include QD () inorganic semiconductor nanocrystals comprising an inorganic core semiconductor material (′) (also referred to as a “core material”) surrounded a shell semiconductor material (″) (also referred to as a “shell material”) having a different band gap (annotated as “core material/shell material”) (e.g. CdS/ZnS). QD () can have a size that typically occurs in the range of 1 nm to 10 nm. QD () can exhibit size-variable emission color due to the quantum confinement effect, where smaller QD () emit at higher energy (lower wavelength) and larger QD () emit at lower energy (higher wavelength) for a given composition. Accordingly, QD () can absorb over a broad range and have photoluminescence emission over a narrow range which can be tuned depending on the material from which the QD () is made and the size of the QD (). Illustrative examples of QD core/shell compositions, and combinations thereof, suitable for use in embodiments of the PENP-MultiDots () can include one or more of: CdS/ZnS, CdSSe/ZnS, CdSe/ZnS, CdTe/ZnS, and CdSeTe/ZnS each having an emission photoluminescence occurring in the range of 420 nm to 880 nm; CuInZnS/ZnS, 540 to 660 nm; and PbS/CdS, 700 nm to 900 nm. However, these examples are not intended to preclude embodiments having other QD () core/shell compositions.

Purified QD () can be bare or can be capped to control QD particle size and/or to prevent QD agglomeration. QD () synthesized by prototypical hot-injection method can be capped with a ligand (), such as, oleylamine and oleic acid after QD purification.H NMR spectroscopy analysis evidence that ligand binding can be highly dynamic, and that oleylamine selectively binds to the surface as oleylammonium bromide in an NC(X)binding motif. Only in the presence of excess oleylamine added after purification does oleic acid bind to the surface, in the form of oleylammonium oleate. Protesescu L, Yakunin S, Bodnarchuk M I, Krieg F, Caputo R, Hendon C H, Yang R X, Walsh A, Kovalenko M V,2015, 15, 3692-3696, hereby incorporated by reference herein. While the use of oleylamine and oleic acid as a capping ligand () is suitable for embodiments of the invention, this is not intended to preclude embodiments using other capping ligand(s) (), as examples, trioctylphosphine oxide, L-histidine, chitosan, polyvinyl alcohol, polyvinylpyrrolidone or combinations thereof.

Typically, bare QD () or capped QD () reach a required level of water solubility and biocompatibility by surrounding the QD () with a P () to produce PENP (). A P () suitable for use with embodiments of the invention can include various embodiments of PS-b-PEG-NHobtained in Block 2I of the synthesis process, above described. Embodiments of PENP () can be produced through the use of various combinations of one or more of: QD (), capping ligand (), and PS-b-PEG-NHhaving PS molecular weights ranging from about 1.5 kDa to about 40 kDa and PEG having molecular weights ranging from about 10 kDa to about 40 kDa.

An illustrative example of a scalable method of production PENP (), including but not necessarily limited to, PENP-MultiDots (), by EHD can include one or more of: constitute PS-b-PEG-NHobtained in, Block 2I at 10 mg/mL into a first 1.5 mL tube. Introduce 240 μl of the desired QD at 5 mg/mL () into a second 1.5 mL tube. Transfer 480 μl of acetone/methanol (60/40) to the QD () in the second 1.5 mL tube. Centrifuge the second 1.5 mL tube containing the QD () at 7000 RCF for 1 min. and then remove supernatant with a 200 μL pipette. Transfer 480 μL of anhydrous tetrahydrofuran (“THF”) to the second 1.5 mL tube containing the QD and mix thoroughly with a pipette. Introduce into a fresh 1.5 mL centrifuge tube 240 μl of THF, 240 μl QD in THE, and 120 μl of the solubilized PS-b-PEG-NHto produce the OP for EHD.

Particular embodiments, EHD can be performed by cleaning the EHD mixing system syringe () three times with THF. Load about 0.6 mL of the OP into the syringe barrel (). Mix the inorganics in the OP thoroughly while loading the syringe (). Attach the syringe () to the syringe pump () and set the syringe pump () to generate a flow rate of the OP containing the inorganics from the syringe needle () in the range of about 11.00 mL/hr. to about 14.00 mL/hr. In particular embodiment the flow rate can be about 12.5 mL/hr. In particular embodiment the flow rate can be about 12.5 mL/hr. Prime the syringe needle () until a drop of the OP forms at the end of the syringe needle (). Twice rinse a 20 mL glass vial () with distilled or deionized water (individually or collectively “DI water”). Introduce about 10 mL of DI water into the 20 mL glass vial. Submerge the syringe needle () into the 20 mL glass vial (). Clean the negative electrode () by submerging in THF, wipe, and rinse with DI water. Place the negative electrode () into the AP contained in the 20 mL glass vial. Place the positive electrode () into the AP contained in the 20 mL glass vial (). In particular embodiments the syringe needle (), if electrically conductive, can act as the positive electrode (). Observe that the positive electrode (), the negative electrode (), and the syringe needle () do not contact. Connect the positive lead () from the voltage source () to the positive electrode () or syringe needle () and connect the negative lead () from the voltage source () to the negative electrode (). Verify that the voltage source () delivers about −1500 V.

The EHD-NP () produced by mixing the OP with the AP under influence of the electrical field can be concentrated using centrifugal filtration. The contents of the 20 mL glass vial () can be transferred to a 100 kDa cutoff centrifugal ultrafiltration column (“CUC”), as an example, SigmaAldrich PN UFC9010D Amicon® Ultra-15 Centrifugal Filter Unit. Centrifuge the CUC at 3000 RCF for 30 min. at about 25° C. (about 77° F.). Transfer 10 mL 50 mM sodium borate, 100 mM sodium phosphate, 7.3-7.5 pH (“borate buffer”) to the CUC. Centrifuge the CUC at 3000 RCF for 30 min. at about 25° C. (about 77° F.). Transfer EHD-NP filtrate from the CUC into a 1.5 mL microcentrifuge tube. Measure and record the volume of the collected EHD-NP filtrate. Transfer 15 μl of EHD-NP filtrate from the 1.5 mL microcentrifuge tube to a 1.5 mL tube and add 285 μl borate buffer. Transfer 290 μl of EHD-NP filtrate to a spectrophotometer cuvette. Measure and record optical density with a spectrophotometer at 450 nm (OD).